N-methyl-d-aspartate andl-aspartate activate distinct receptors in piriform cortex

The effects of ionophoretically applied N-methyl-DL-aspartate (NMDA) and aspartate on identified pyramidal neurons in rat piriform cortex were examined in isolated, submerged, and perfused brain slices. NMDA was more potent than aspartate in eliciting neuronal discharge. Perfusion of the acidic amino acid antagonists, DL-2-amino-5-phosphonovalerate (APV), 10(-6) or 10(-5) M, DL-2-amino-7-phosphonoheptanoate (APH), 10(-5) M, and gamma-D-glutamylglycine (gamma DGG), 10(-5) M, selectively blocked the response to NMDA without effect on the response to aspartate. At higher concentrations which blocked responses to both NMDA and aspartate, gamma DGG blocked kainate responses and depressed glutamate and quisqualate responses. These results suggest that in piriform neurons NMDA and aspartate act at distinct receptor sites, not a common receptor site, and that both of these sites are distinct from those that mediate responses to glutamate, quisqualate, and kainate.     

Apoptotic Cell Death and Caspase-3 Activation Induced by N-Methyl-d-Aspartate Receptor Antagonists and Their Prevention by Insulin-Like Growth Factor I

The effect of N-methyl-D-aspartate (NMDA) receptor antagonists on cell viability was studied in rat primary cortical cells. NMDA antagonists [MK-801 and 2-amino-5-phosphonovalerate (APV)] induced cell shrinkage, nuclear condensation or fragmentation, and internucleosomal DNA fragmentation. Treatment of cells with MK-801 (an NMDA antagonist) for 1-2 days induced apoptotic cell death in a dose-dependent manner (1 nM to 10 microM). NMDA (25 microM), however, inhibited the MK-801 (0.1 microM)-induced apoptotic cell death. MK-801 and APV decreased the concentration of intracellular calcium ion. Activation of caspase-3 was accompanied by MK-801-induced cell death in a dose-dependent manner, and an inhibitor of caspase-3 reduced the cell death. Further, cycloheximide (0.2 microg/ml) completely protected the cells from MK-801-induced apoptotic cell death and caspase-3 activation. Insulin-like growth factor I completely attenuated MK-801-induced apoptotic cell death and caspase-3 activation. These results demonstrated that the moderate NMDA receptor activation is probably involved in the survival signal of the neuron.     

Effect of caerulein on decreased latency of passive avoidance response in rats treated with NMDA receptor antagonists

The effect of subcutaneous injection of caerulein on memory impairment induced by intracerebroventricular administration of NMDA receptor antagonists was examined in the passive avoidance response of the rat. When rats were treated with AP5, AP7, CPP or MK-801, the retention latencies decreased markedly. However, in rats that received caerulein immediately after the training trials, the latency increased to some extent. Pretreatment with caerulein and subsequent injection of the competitive NMDA receptor antagonists AP5, AP7 and CPP caused a more apparent increase in the latency. The noncompetitive NMDA receptor antagonist MK-801 was not affected by pretreatment with caerulein. The difference might be, at least in part, due to the sites of action of these NMDA receptor antagonists.     

Modulation of the Mesolimbic Dopamine System by Glutamate

Glutamate has been shown to modulate motor behavior, probably via N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that are involved in the control of the mesolimbic dopamine (DA) system, that is, the ventral tegmental area (VTA)-nucleus accumbens (NAC). In the present study, we investigated the effects of uncompetitive (MK-801) and competitive [DL-2-amino-5-phosphonopentanoic acid (AP-5), CGP 40116] NMDA receptor antagonists and NMDA and AMPA on DA release in the mesolimbic system and on motor behavior. Systemic injection and intrategmental infusion of MK-801 increased DA levels in the VTA, but the systemic administration enhanced DA exclusively in the NAC and increased motor behavior. In contrast, intrategmental infusion of AP-5, but not the systemic administration of its lipophilic analogue CGP 40116, decreased the DA release in the two regions without affecting motor behavior. NMDA and AMPA infusion into the VTA increased DA levels in both areas. This increase was accompanied by a strong motor behavioral stimulation after NMDA but only a moderate increase after AMPA infusion. The present results indicate that mesolimbic DA neurons are controlled by the glutamatergic system and that the effects of uncompetitive and competitive NMDA receptor antagonists on DA release are mediated by an interaction with different brain areas. These findings may account for the different effects of NMDA receptor ligands on motor behavior.     

Identification of a Novel N-Methyl-D-Aspartate Receptor Population in the Rat Medial Thalamus

To evaluate the possibility of pharmacologically distinct N-methyl-D-aspartate (NMDA) receptor subtypes, quantitative autoradiography was used to determine the potency of several compounds as inhibitors of L-[3H]glutamate or [3H]MK-801 binding to rat brain NMDA receptors in 10 brain regions. Competitive NMDA receptor antagonists displayed differing pharmacological profiles in the forebrain, cerebellum, and medial regions of the thalamus (midline nuclei). For example, compared with other competitive antagonists, 3-[(+/-)-2-carboxypiperazin-4-yl]propyl-1-phosphonate (CPP) and LY-233536 were especially weak displacers of L-[3H]glutamate binding in the cerebellum. In the the medial thalamus, CPP and D-2-amino-5-phosphonopentanoate displayed relatively low affinities, whereas LY-233536 was relatively potent. The noncompetitive NMDA receptor antagonists also displayed regional variations in their pharmacological profiles. Relative to other regions, [3H]MK-801 binding in the cerebellum was weakly displaced by MK-801 and potently displaced by dextromethorphan and SKF-10047. In the medial thalamus, 1-[1-(2-thienyl)-cyclohexyl]piperidine was relatively potent and SKF-10047 was relatively weak. These results confirm previous suggestions that the cerebellum contains a distinct NMDA receptor subtype and indicate that nuclei of the medial thalamus contain a novel NMDA receptor subtype that is distinct from both those found in the cerebellum and in the forebrain.     

Competitive and non-competitive N-methyl-D-aspartate antagonists fail to prevent the induction of methamphetamine-induced sensitization.

In order to elucidate the possible roles of the glutamate system in the mechanisms underlying behavioral sensitization, which is used as an animal model for human psychosis, we investigated the effects of 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and MK-801 ((+)-dizocilpine), a competitive and noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, respectively, on methamphetamine-induced behavioral sensitization in rats. Administration of 0.5 mg/kg MK-801 enhanced 2 mg/kg methamphetamine-induced hyperactivity, whereas it reduced 6 mg/kg methamphetamine-induced stereotyped behavior markedly. CPP (10 mg/kg) reduced 2 mg/kg methamphetamine-induced stereotypy slightly. Repeated treatment with 2 and 6 mg/kg methamphetamine alone induced progressive augmentation of stereotypy, whereas combining either MK-801 or CPP with methamphetamine treatment abolished or attenuated this augmentation. However, when rats were challenged with methamphetamine after a 7-day period of abstinence, the intensity of stereotypy among the rats pretreated with repeated doses of methamphetamine alone or in combination with MK-801 or CPP did not differ significantly. These results indicate that competitive and non-competitive NMDA receptor antagonists modulate acute methamphetamine-induced abnormal behavior and sensitization expression, but they failed to prevent the induction of the neural mechanisms underlying behavioral sensitization.     

Use of a conflict procedure in pigeons to characterize anxiolytic drug activity: evaluation of N-methyl-D-aspartate antagonists.

Because of its apparent effectiveness in detecting non-benzodiazepine anxiolytic agents, a recently introduced conflict procedure in pigeons was used to evaluate possible anti-punishment activity of various N-methyl-d-aspartate (NMDA) antagonists. Punished responding was significantly increased by competitive NMDA antagonists (CPP, CGS 19755), but not by noncompetitive NMDA antagonists acting at either the ion channel (PCP, ketamine, MK-801), the glycine site (kynurenic acid, 7-chlorokynurenic acid, ACPC), or the polyamine site (ifenprodil) of the NMDA receptor complex; the proposed glutamate antagonist, riluzole, was also ineffective.     

NMDA receptor antagonism disrupts the development of morphine analgesic tolerance in male, but not female C57BL/6J mice

Multiple studies demonstrate that coadministration of N-methyl-D-aspartate (NMDA) receptor antagonists with the opioid agonist morphine attenuates the development of analgesic tolerance. Sex differences in the effects of noncompetitive, but not competitive NMDA receptor antagonists on acute morphine analgesia, have been reported in mice, yet the role of sex in modulation of morphine tolerance by NMDA receptor antagonists has yet to be addressed. Therefore, we tested whether there is a sex difference in the effect of NMDA receptor antagonists on the development of morphine analgesic tolerance in C57BL/6J mice. Acutely, at a dose required to affect morphine tolerance in male mice, the noncompetitive NMDA receptor antagonist dizocilpine (MK-801) prolonged morphine analgesia similarly in both sexes in the hot plate and tail withdrawal assays. In the hot plate assay, coadministration of MK-801 or the competitive antagonist 3-(2-carboxpiperazin-4-yl)propyl-1-phosphanoic acid (CPP) with morphine attenuated the development of tolerance in male mice, while having no effect in females. Like normal and sham females, ovariectomized mice were similarly insensitive to the attenuation of morphine tolerance by MK-801 in the hot plate assay. Surprisingly, in the tail withdrawal assay, MK-801 facilitated the development of morphine-induced hyperalgesia and tolerance in males but not females. The results demonstrate that male mice are more sensitive to modulation of nociception and morphine analgesia after repeated coadministration of NMDA receptor antagonists. Furthermore, the underlying mechanisms are likely to be different from those mediating the sex difference in the modulation of acute morphine analgesia that has previously been reported.     

Glutathione Is an Endogenous Ligand of Rat Brain N-Methyl-D-Aspartate (NMDA) and 2-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionate (AMPA) Receptors

A study was made of the effects of reduced (GSH) and oxidized (GSSG) glutathione on the Na⁺-independent and N-methyl-D-aspartate (NMDA) displaceable bindings of glutamate, on the binding of kainate, 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and ligands of the brain NMDA receptor-ionophore complex: glycine, dizocilpine (MK-801) and (±)-3-(2-car-boxypiperazin-4-yl)propyl-1-phosphonate (CPP). GSH and GSSG strongly inhibited the binding of glutamate, CPP and AMPA, kainate and glycine binding being less affected. Both peptides enhanced the binding of dizocilpine in a time- and concentration-dependent manner. This activatory effect was not additive to that of saturating concentrations of glutamate or glutamate plus glycine. The activation of dizocilpine binding by GSH and GSSG was prevented by the competitive NMDA and glycine antagonists DL-2-amino-5-phosphonovalerate and 7-chlorokynurenate. GSH and GSSG may be endogenous ligands of AMPA and NMDA receptors, binding preferably to the glutamate recognition site via their -glutamyl moieties. In addition to this, at millimolar concentrations they may regulate the redox state of the NMDA receptor-ionophore complex.     

Conserved structural and functional control of N-methyl-D-aspartate receptor gating by transmembrane domain M3

The molecular events controlling glutamate receptor ion channel gating are complex. The movement of transmembrane domain M3 within N-methyl-d-aspartate (NMDA) receptor subunits has been suggested to be one structural determinant linking agonist binding to channel gating. Here we report that covalent modification of NR1-A652C or the analogous mutation in NR2A, -2B, -2C, or -2D by methanethiosulfonate ethylammonium (MT-SEA) occurs only in the presence of glutamate and glycine, and that modification potentiates recombinant NMDA receptor currents. The modified channels remain open even after removing glutamate and glycine from the external solution. The degree of potentiation depends on the identity of the NR2 subunit (NR2A < NR2B < NR2C,D) inversely correlating with previous measurements of channel open probability. MTSEA-induced modification of channels is associated with increased glutamate potency, increased mean single-channel open time, and slightly decreased channel conductance. Modified channels are insensitive to the competitive antagonists D-2-amino-5-phosphonovaleric acid (APV) and 7-Cl-kynurenic acid, as well as allosteric modulators of gating (extracellular protons and Zn(2+)). However, channels remain fully sensitive to Mg(2+) blockade and partially sensitive to pore block by (+)MK-801, (-)MK-801, ketamine, memantine, amantadine, and dextrorphan. The partial sensitivity to (+)MK-801 may reflect its ability to stimulate agonist unbinding from MT-SEA-modified receptors. In summary, these data suggest that the SYTANLAAF motif within M3 is a conserved and critical determinant of channel gating in all NMDA receptors.     

Pentobarbital-like effects of N-methyl-D-aspartate antagonists in mice

The effects of the competitive N-methyl-D-aspartate (NMDA) antagonist, 3-[(+/-)-2-carboxypiperazin-4-yl]propyl-1-phosphonic acid (CPP), and of the noncompetitive NMDA antagonist, dizocilpine (MK-801), were determined in mice trained to discriminate pentobarbital (20 mg/kg i.p.) from saline under a standard two-lever fixed-ratio 20 schedule of sweetened milk reinforcement. CPP substituted for pentobarbital; however, pentobarbital-lever responding was usually associated with decreases in response rates. Dizocilpine produced a maximum average of only 62% pentobarbital-lever responding, accompanied by a 50% decrease in response rates. These results suggest that pentobarbital-like discriminative stimulus effects are more likely to be produced by competitive than by noncompetitive NMDA antagonists. This extends previous observation in rats and provides further evidence for differences in the behavioral effects of competitive and noncompetitive NMDA antagonists and for an overlap in the behavioral pharmacology of NMDA antagonists and classical CNS depressants.     

Role of Excitatory Amino Acid Receptors in K+-and Glutamate-Evoked Release of Endogenous Adenosine from Rat Cortical Slices

K+ and glutamate released endogenous adenosine from superfused slices of rat parietal cortex. The absence of Ca2+ markedly diminished K+- but not glutamate-evoked adenosine release. Tetrodotoxin decreased K+- and glutamate-evoked adenosine release by 40 and 20%, respectively, indicating that release was mediated in part by propagated action potentials in the slices. Inhibition of ecto-5'-nucleotidase by alpha,beta-methylene ADP and GMP decreased basal release of adenosine by 40%, indicating that part of the adenosine was derived from the extracellular metabolism of released nucleotide. In contrast, inhibition of ecto-5'-nucleotidase did not affect release evoked by K+ or glutamate, suggesting that adenosine was released as such. Inhibition of glutamate uptake by dihydrokainate potentiated glutamate-evoked release of adenosine. Glutamate-evoked adenosine release was diminished 50 and 55% by the N-methyl-D-aspartate (NMDA) receptor antagonists, DL-2-amino-5-phosphonovaleric acid and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801), respectively. The remaining release in the presence of MK-801 was diminished a further 66% by the non-NMDA receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione, suggesting that both NMDA and non-NMDA receptors were involved in glutamate-evoked adenosine release. Surprisingly, K+-evoked adenosine release was also diminished about 30% by NMDA antagonists, suggesting that K+-evoked adenosine release may be partly mediated indirectly through the release of an excitatory amino acid acting at NMDA receptors.     

Effects of ifenprodil on the N-methyl-D-aspartate receptor ionophore complex in rat brain.

The effects of a cerebral anti-ischemic drug ifenprodil on the receptor ionophore complex of an N-methyl-D-aspartate (NMDA)-sensitive subclass of central excitatory amino acid receptors were examined using [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10- imine (MK-801) binding in rat brain synaptic membrane preparations as a biochemical measure. The binding in membrane preparations not extensively washed was markedly inhibited not only by competitive NMDA antagonists such as (+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic, D-2-amino-5-phosphonovaleric and D-2-amino-7-phosphonoheptanoic acids, but also by competitive antagonists at the strychnine-insensitive glycine (Gly) site including 7-chlorokynurenic acid and 6,7-dichloroquinoxaline-2,3-dione. Among several proposed ligands for alpha-adrenergic receptors tested, ifenprodil most potently inhibited the binding in these membrane preparations due to a decrease in the density of the binding sites without significantly affecting the affinity. Ifenprodil also inhibited the binding of [3H]N-[1-(2-thienyl)cyclohexyl]piperidine as well as of [3H]MK-801 to open NMDA channels in a concentration-dependent manner at concentrations above 10 nM in membrane preparations extensively washed but not treated by a detergent, with a Hill coefficient of less than unity. Further treatment of extensively washed membrane preparations with a low concentration of Triton X-100 resulted in an almost complete abolition of [3H]MK-801 binding, and the binding was restored to the level found in membrane preparations not extensively washed following the addition of both L-glutamic acid (Glu) and Gly. Ifenprodil was effective in inhibiting [3H]MK-801 binding via reducing both initial association and dissociation rates in Triton-treated membrane preparations, irrespective of the presence of Glu and Gly added. The binding in Triton-treated membrane preparations was additionally potentiated by the polyamine spermidine in a concentration-dependent manner at concentrations above 10 microM in the presence of both Glu and Gly at maximally effective concentrations. Ifenprodil invariably diminished the abilities of these three stimulants to potentiate [3H]MK-801 binding at concentrations over 1 microM in a manner that the maximal responses each were reduced. These results suggest that ifenprodil does not interfere with the NMDA receptor complex as a specific isosteric antagonist at the polyamine domain in contrast to the prevailing view.     

[3H]MK-801 Labels a Site on the N-Methyl-D-Aspartate Receptor Channel Complex in Rat Brain Membranes

The potent noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist [3H]MK-801 bound with nanomolar affinity to rat brain membranes in a reversible, saturable, and stereospecific manner. The affinity of [3H]MK-801 was considerably higher in 5 mM Tris-HCl (pH 7.4) than in previous studies using Krebs-Henseleit buffer. [3H]MK-801 labels a homogeneous population of sites in rat cerebral cortical membranes with KD of 6.3 nM and Bmax of 2.37 pmol/mg of protein. This binding was unevenly distributed among brain regions, with hippocampus greater than cortex greater than olfactory bulb = striatum greater than medulla-pons, and the cerebellum failing to show significant binding. Detailed pharmacological characterization indicated [3H]MK-801 binding to a site which was competitively and potently inhibited by known noncompetitive NMDA receptor antagonists, such as phencyclidine, thienylcyclohexylpiperidine (TCP), ketamine, N-allylnormetazocine (SKF 10,047), cyclazocine, and etoxadrol, a specificity similar to sites labelled by [3H]TCP. These sites were distinct from the high-affinity sites labelled by the sigma receptor ligand (+)-[3H]SKF 10,047. [3H]MK-801 binding was allosterically modulated by the endogenous NMDA receptor antagonist Mg2+ and by other active divalent cations. These data suggest that [3H]MK-801 labels a high-affinity site on the NMDA receptor channel complex, distinct from the NMDA recognition site, which is responsible for the blocking action of MK-801 and other noncompetitive NMDA receptor antagonists.     

Interaction of NMDA and Dopamine D2L Receptors in Human Neuroblastoma SH-SY5Y Cells

Abstract: To understand the mechanism of interaction of the dopamine D_2L receptors with NMDA receptors, we have developed a model by transfecting human neuroblastoma SH-SY5Y cells with the human dopamine D_2L receptor gene. In vitro blockade of NMDA receptors by the specific antagonists MK-801 and (±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) on human neuroblastoma SH-SY5Y cells expressing human dopamine D_2L receptors resulted in a significant increase in the density of D_2L receptors without a significant change in receptor affinity. Moreover, the dopamine receptor mRNA level increased by ∼50% by the blockade of NMDA with MK-801. These results suggest a possible interaction of NMDA and dopamine D_2L receptors in neuroblastoma SH-SY5Y cells. This system would serve as an excellent model to study the molecular mechanisms involved in the interaction of these two receptors.     

N-Methyl-D-Aspartate receptors in acquisition of the short-term memory in the honeybeeApis mellifera

Role of NMDA receptors in the process of associative learning has been studied in the honeybeeApis mellifera L. in behavior experiments, using method of conditional reflexes. To determine pharmacological profile of NMDA receptors, effects of Mg²⁺ ions, NMDA, glycine (Gly), antagonist of the glycine site 5,7-dichlorokynurenic acid (DCK), competitive antagonists of NMDA receptors: D,L-2-aminophosphovalerate (APV), L-2-aminophosphobutyrate (APB), and D-glutamyl-aminomethylphosphonic acid (GAMP) as well of antagonists of NMDA receptor ion channels MK-801 and ketamine (Ket) were studied on acquisition of alimentary conditional reflex and its retention in memory. NMDA increased capacity for learning by stimulating shortterm memory. The NMDA receptor co-agonist Gly activated this NMDA effect. DCK eliminated the NMDA and Gly stimulatoty effects. All tested antagonists at millimolar concentrations inhibited associative function. The data obtained confirm our hypothesis about participation of NMDA receptors in processes of formation of short-term memory in the honeybeeApis mellifera and suggest that functional characteristics of the NMDA receptors involved in the process of associative learning in the honeybee resemble those in mammals.     

Mode of binding of [3H]dibenzocycloalkenimine (MK-801) to the N-methyl-D-aspartate (NMDA) receptor and its therapeutic implication

Binding of the labeled anticonvulsant drug [³H]dibenzocycloalkenimine (³H]MK-801) to the N-methyl-D-aspartate (NMDA) receptor and its dissociation from the receptor at 25°C are slow processes, both of which follow first order kinetics (t_1/270 and 180 min, respectively). Both reactions are markedly accelerated by glutamate and glycine (t_1/22-8 and 4 min, respectively), which allow bimolecular association kinetics of the labeled drug with the receptors whereas equilibrium binding of [³H]MK-801 (K_d 2–4 nM) is hardly affected by glutamate and glycine. The data suggest that MK-801 acts as a steric blocker of the NMDA receptor channel. The competitive antagonist D-(−)-2-amino-5-phosphovaleric acid (AP-5) freezes the receptor in a state which precludes either binding of [³H]MK-801 to the receptor channel or its dissociation from it. These findings have therapeutic implications.     

Excitotoxic mechanism of cell swelling in rat cerebral cortical slices treated acutely with ammonia

Swelling of CNS cells due to endogenous ammonia is a major cause of cerebral oedema in hyperammonaemic encephalopathies. In the present study, incubation in the presence of 5mM ammonium acetate ("ammonia") decreased steady-state distribution of [14C]inulin within incubated rat cerebrocortical minislices, indicating cell swelling. NMDA receptor antagonists, MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d]cycloheptene-5,10-imine maleate,10 microM) and DL-AP-5 (DL-2-amino-5-phosphonovaleric acid, 250 microM), a nitric oxide synthase inhibitor, L-nitroarginine (L-NNA, 500 microM), and an antioxidant, taurine (Tau, 10 mM), markedly attenuated the cell volume-increasing effect of ammonia. The effect of Tau (10mM) was abolished by the GABA(A) receptor antagonist bicuculline (100 microM), but was unaffected by the Tau transport inhibitor guanidynoethyl-sulfonate (GES, 500 microM). Ammonia increased the slice content of Gln, an amino acid whose excess accumulation has been implicated in hyperammonemic oedema. However, treatments that reduced the cell volume did not affect Gln content. These results indicate that ammonia-induced cell swelling is in a large degree mediated by overactivation of NMDA receptors and the ensuing generation of NO and free radicals.     

Solubilization of Spermidine-Sensitive (+)-[3H]5-Methyl-10,11 -Dihydro-5H-Dibenzo[a,d]cyclohepten-5,10-Imine ([3H]MK-801) Binding Activity from Rat Brain

The receptor-ionophore complex of the N-methyl-D-aspartate (NMDA)-sensitive receptor was solubilized by deoxycholic acid from rat brain using (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne ([3H]MK-801) binding as a marker for the receptor. Gel filtration of the solubilized preparations on a Sephadex G-25 column revealed significant [3H]MK-801 binding sensitive to potentiation by glutamate and glutamate/glycine, which was prevented by competitive antagonists for the NMDA and strychnine-insensitive glycine (GlyB) sites. In contrast to NMDA and glycine, spermidine markedly potentiated the amount of [3H]MK-801 binding in solubilized preparations by increasing the apparent affinity of the ligand. In the presence of all three stimulants, the solubilized preparations exhibited pharmacological profiles similar to those in the membrane preparations. These results clearly indicate that the whole macromolecular NMDA receptor-ionophore complex is solubilized under the experimental conditions used.